Background
This study was aimed to investigate the role and specific molecular mechanism of HIF1A-AS2/miR-665/IL6 axis in regulating osteogenic differentiation of adipose-derived stem cells (ASCs) via the PI3K/Akt signaling pathway.
Conclusions
LncRNA HIF1A-AS2 could sponge miR-665 and hence upregulate IL6, activate the PI3K/Akt signaling pathway, and ultimately promote ASC osteogenic differentiation.
Methods
RNAs' expression profile in normal/osteogenic differentiation-induced ASCs (osteogenic group) was from the Gene Expression Omnibus database. The analysis was carried out using Bioconductor of R. Gene Set Enrichment Analysis and Kyoto Encyclopedia of Genes and Genomes dataset were applied to identify up- and downregulated signaling pathways. Co-expression network of specific lncRNAs and mRNAs was structured by Cytoscape, while binding sites amongst lncRNA, mRNA, and miRNA were predicted by TargetScan and miRanda. ASCs were derived from human adipose tissue and were authenticated by flow cytometry. ASC cell function was surveyed by alizarin red and alkaline phosphatase (ALP) staining. Molecular mechanism of HIF1A-AS2/miR-665/IL6 axis was investigated by RNAi, cell transfection, western blot, and qRT-PCR. RNA target relationships were validated by dual-luciferase assay.
Results
HIF1A-AS2 and IL6 were highly expressed while miR-665 was lowly expressed in induced ASCs. HIF1A-AS2 and IL6 improved the expression level of osteoblast markers Runx2, Osterix, and Osteocalcin and also accelerated the formation of calcium nodule and ALP activity, yet miR-665 had opposite effects. HIF1A-AS2 directly targeted miR-665, whereas miR-665 repressed IL6 expression. Moreover, the HIF1A-AS2/miR-665/IL6 regulating axis activated the PI3K/Akt signaling pathway. Conclusions: LncRNA HIF1A-AS2 could sponge miR-665 and hence upregulate IL6, activate the PI3K/Akt signaling pathway, and ultimately promote ASC osteogenic differentiation.